Proteome-wide analysis of cysteine oxidation using Stable Isotope Cysteine Labelling with Iodoacetamide (SICyLIA)

Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules that act through the oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Identifying cysteine oxidation on a whole-proteome scale remains a technical challenge due to the low abundance of oxidised thiols. Redox proteomics techniques therefore use multistep enrichment protocols, but these have inherent limitations and inform only on the enriched proteome. We developed stable isotope cysteine labelling with iodoacetamide (SICyLIA), a simple, unbiased, and robust mass spectrometry-based workflow for thiol oxidation analysis. SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity. We applied SICyLIA to diverse cellular models and primary tissues and generated the most in-depth thiol oxidation profiles to date. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys showed oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies.

with iodoacetamide (SICyLIA), a simple, unbiased, and robust mass spectrometry-based workflow for thiol oxidation analysis. SICyLIA does not require enrichment steps and achieves unbiased proteomewide sensitivity. We applied SICyLIA to diverse cellular models and primary tissues and generated the most in-depth thiol oxidation profiles to date. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys showed oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies.  • EASY-nLC II 1200 nanoscale C18 reverse-phase liquid chromatography (Thermo Scientific)
(2) Pre-chill bench top centrifuge to 4 °C (3) Prepare lysis buffer 1 (100 mM Tris-HCl pH 7.5, 4% SDS) and immediately before cell lysis and protein extraction, add light or heavy iodoacetamide (IAM) to lysis buffer 1 to achieve 55 mM IAM solutions. → Note: iodoacetamide is unstable and light sensitive, so solutions are best made fresh and kept in the dark until use.
(4) Remove medium and wash cell monolayers twice with pre-chilled PBS (4 °C) ensuring to aspirate PBS thoroughly.
(5) Add 500 µl lysis buffer 1 with IAM per dish and immediately scrape cells using a cell lifter.
(7) Sonicate lysates for 4 x 5 s to shear DNA/RNA. → Note: heavy IAM ( 13 C 2 D 2 H 2 INO) can be affected by hydrogen-deuterium exchange, which is exacerbated at high pH and temperature. This has been minimised in our protocol through optimisation of pH and temperature at all steps. Here, it is important to use ice during sonication to ensure samples do not heat up, yet prevent the SDS buffer from precipitating on ice over time. Clean sonicator thoroughly with 70% EtOH between samples to prevent cross-contamination.
(9) Transfer supernatants to new Eppendorf tubes and incubate in a bench top shaker at 1400 rpm for 1 h in the dark at room temperature (RT).
(10) Determine protein concentration of samples using bicinchoninic acid (BCA) assay or equivalent.

Tissue-based SICyLIA application
Note: this procedure was optimised for the analysis of mouse kidney tissues. Tissue resection and homogenisation strategy suitable for the tissue type under study may need to be optimised.
(1) Pre-chill plastic or glass dishes, scalpel, and forceps on dry ice.
(2) Pre-chill Precellys24 bead-based homogeniser (Bertin Instruments, or equivalent). 6 (3) Prepare lysis buffer 1 and immediately before tissue homogenisation, add light or heavy IAM to lysis buffer 1 to achieve 55 mM IAM solutions. → Note: iodoacetamide is unstable and light sensitive, so solutions are best made fresh and kept in the dark until use.
(4) Prepare homogenisation tubes by adding 800 µl lysis buffer 1 with IAM to the ceramic beads.
(5) Sacrifice mice by cervical dislocation. → Note: this is preferred over CO 2 inhalation, as this can induce tissue hypoxia and influence cellular redox status.
(6) Excise kidneys, place in plastic tubes, and snap freeze tubes in liquid nitrogen.
(7) Once frozen, place kidneys on pre-chilled plastic or glass dishes on dry ice and excise representative samples. → Note: ensure tissue does not defrost at any stage of the procedure until homogenisation to preserve cellular redox status.
(8) Add frozen tissue slices to homogenisation tubes with lysis buffer, and immediately homogenise for 3 × 20 s at 5000 rpm.
(10) Transfer supernatants to new Eppendorf tubes and incubate in a bench top shaker at 1400 rpm for 1 h in the dark at RT.
(11) Determine protein concentration of samples using bicinchoninic acid (BCA) assay or equivalent.

Part 2. Reduce/alkylate and proteome digestion
(1) Pre-chill bench top shaker and centrifuge to 4 °C   → Note: proteome digestion is usually carried out at higher pH and temperature to allow the enzymes to work optimally, but this can also promote hydrogen-deuterium exchange on heavy IAM. We established that overnight digestion at RT and pH 7.0 does not compromise efficiency as it had minimal effects on the miscleavage rate, while minimising hydrogen-deuterium exchange. Proceed with the samples intended for proteome normalisation to Part 3.

Part 3. Dimethyl labelling for proteome normalisation
We followed the on-column protocol described by Boersema and colleagues [1].
(3) Load the samples onto their respective columns.
(5) Label peptides with light and heavy formaldehyde/cyanoborohydride solutions, using 5 x 1 ml each.
(9) Mix the heavy and light labelled samples using the same label-swap replication approach as for SICyLIA samples (i.e. heavy dimethylated wild-type replicate 1 with light dimethylated knock-out replicate 1 forms forward replicate 1; light dimethylated labelled wild-type replicate 2 with heavy 9 dimethylated knock-out replicate 2 forms reverse replicate 1, etc.

Part 4. Off-line reverse phase HPLC fractionation
(1) Reduce all sample volumes to 300 µl using vacuum centrifugation to remove ACN and TFA.
(2) Bring the volume up to 500 µl per sample to match the injection loop volume, using HPLC solvent A.
(4) Inject samples (500 µl) manually through a Rheodyne valve onto the RP-HPLC column. (7) Dry the fractions to completion using vacuum centrifugation and store at -80 °C until analysis.
(2) Separate samples by nanoscale C18 reverse-phase liquid chromatography using an EASY-nLC II 1200 (Thermo Scientific) (3) Elute using a binary gradient with MS solvent A (2% acetonitrile, 0.1% formic acid in water) and MS solvent B (80% acetonitrile, 0.1% formic acid in water) at a flow rate of 300 nl/min using different gradients, which were optimised for three sets of fractions: 1-7, 8-15, and 16-21. For all gradients, use 20 min for step one and 7 min for step two. Change the percentage of MS solvent B (%B) as follows: For F1-7, %B was 2 at the start, 20 at step one, and 39 at step two. For F8-14, %B was 4 at the start, 23 at step one, and 43 at step two. For F15-21, %B was 6 at the start, 28 at step one, and 48 at step two. Follow all gradients by a washing step (100% B) for 10 min followed by a 5 min re-equilibration step (5%), for a total run time of 40 min.
Eluting peptides are electrosprayed into the mass spectrometer (Q-Exactive HF, Thermo Scientific) using a nanoelectrospray ion source (Thermo Scientific). An Active Background Ion Reduction Device is used to decrease air contaminants signal level.

Part 5. Data analysis
We used MaxQuant version 1.5.5.1 [2] and searched with Andromeda search engine [3]. The default parameters were used with modifications as specified below.

MaxQuant data processing
(1) Perform first and main searches with precursor mass tolerances of 20 ppm and 4.5 ppm, respectively, and MS/MS tolerance of 20 ppm.
(2) Set minimum peptide length to six amino acids and require specificity for trypsin cleavage, allowing up to two missed cleavage sites.
(3) Require at least one uniquely assigned peptide and a minimum ratio count of 2 for a protein to be quantified.
(4) Require that only unique peptides are used for protein quantification.
(5) Specify methionine oxidation and N-terminal acetylation as variable modifications.
(7) Set modification by light and heavy iodoacetamide on cysteine residues (carbamidomethylation) as label type modification in Andromeda configuration with composition sets HNOCx(2)Hx(2) for heavy and H(3)NOC(2) for light label.
(9) Process both data sets (iodoacetamide heavy/light and dimethyl heavy/light) at the same time in MaxQuant using different parameters, by defining these with the Parameter Groups option.
(10) Quantitation of cysteine oxidation reported in the MaxQuant output peptide.txt file, and quantification of proteins reported in the proteinGroups.txt file, will be used for further analysis. → Note: for all the other setting we kept the default MaxQuant parameters.
(6) Processing ---Transform "1/(x)" the columns "Ratio H/L normalized" of the dimethyl-labelled reverse replicates. → Note: this ensures the ratio values of all replicates now follow the format "wild-type over knockout".

Part 2. Peptide analysis
(1) Import the peptides.txt and pre-processed proteinGroups.txt files into Perseus (2) Multi processing ---Matching rows by name. Match "id" in peptides.txt with "Peptide IDs" in proteinGroups.txt file and import the pre-processed "Ratio H/L normalized" columns of the dimethyllabelled replicates.
(5) Processing ---Filter rows based on categorical column "Unique (Groups)". Remove matching rows with value "no", reduce matrix. → Note: with step (5) we ensure to keep only those peptides that are unique to a single protein group in the proteinGroups file.
(8) Processing ---Transform "1/(x)" the columns "Ratio H/L normalized" of the iodoacetamide-labelled reverse replicates. → Note: this has already been done for the protein ratio values in Part 1 step (6), so only transform the peptide ratio values here. This ensures the ratio values of all replicates now follow the format "wild-type over knock-out".
(11) Processing ---Combine main columns using Operation "x/y" to divide the ratio values of each peptide by the ratio values of the parent protein. This gives the normalised peptide oxidation ratio for each peptide.
(13) Processing ---Categorical annotation rows. Action: Create Group1, include the normalised peptide oxidation ratios for all replicates of each experimental condition.
(15) Processing ---Average groups. Grouping "Group1", Average type "Median", Keep original data, Add "Standard deviation". → The median and its standard deviation will be used for further data analysis and interpretation.
Therefore, it is important to define the minimum number of valid values for inclusion here based on the number of replicate experiments used (i.e. peptides must be quantified in at least 3 out of 4 replicate experiments).